The absorption of a photon by rhodopsin in vertebrate rods initiates a series of biochemical events leading to a transient decrease in the light- sensitive outer segment membrane current by modulation of the cGMP concentration. Among the steps determining the light-sensitive current kinetics and light sensitivity is activation of a cGMP-phosphodiesterase, whose activator protein, G protein, is in turn catalytically activated by photoisomerized rhodopsin. Rhodopsin has been proposed to be inactivated by phosphorylation by a rhodopsin kinase. Experiments are proposed here to determine the rate limiting steps(s) in the light-activation of G protein and cGMP-phosphodiesterase. The hypotheses (1) that activation of G protein and of PDE occurs solely via membrane diffusion and (2) that G protein or PDE activation requires an aqueous diffusion step will be tested. The gain of the G protein and PDE activation steps will be measured and the biochemical mechanisms that determine or limit the gain will be examined. The GTPase is an inactivation mechanism of light-activated PDE will be tested. The hypothesis that rhodopsin phosphorylation is sufficient to inactivate the catalytic form of photoisomerized rhodopsin will be tested. The experiments rely upon four methodologies: (1) pH electrode measurements of cGMP phosphodiesterase (PDE) activity in toad and bovine rod disk membranes (RDM); (2) measurement of sigma32P GTP binding to toad and bovine RDM; (3) measurement of light-activated phosphorylation in RDM and (4) reconstitution of purified PDE and G protein into bovine RDM stripped of peripheral proteins.